The long-term goal of this project is to identify and develop drugs that induce mitochondrial biogenesis. A variety of diverse acute insults such as ischemia/reperfusion (I/R), drug, toxicant, and trauma lead to mitochondrial dysfunction and results in cell injury and death in many organs (e.g. heart, brain, liver and kidney). Furthermore, mitochondrial dysfunction is a component of many chronic diseases such as metabolic syndrome and neurodegeneration, and aging. There are no effective therapies that can restore mitochondrial function and, thus, a drug that produces mitochondrial biogenesis may have extensive utility in the treatment of a broad group of diseases. Mitochondrial biogenesis drug development has been hindered by poor cellular models with limited mitochondrial function, surrogate markers of biogenesis that have high false-positive and false negative-rates, and no moderate to high throughput screening assay to measure functional mitochondrial biogenesis. We have studied mitochondrial biogenesis for the past five years in primary cultures of renal proximal tubular cells (RPTC) that maintain in vivo like mitochondrial function. We recently adapted and optimized our RPTC system to the 96-well Seahorse Biosciences respirometer and validated the assay using chemicals known to induce mitochondrial biogenesis. It was found that the maximum FCCP-uncoupled rates in these cells are limited only by the number of functional mitochondria and, thus, increases in the FCCP- uncoupled rates are functional biomarkers of mitochondrial biogenesis. Using this assay we screened a test library of 500 compounds randomly selected from the DIVERSet 50,000-compound library. We identified 7 hits that were confirmed with secondary assays of increased expression of mitochondrial proteins, the "gold standard". Thus, we can start immediately to use our phenotypic assay with compound libraries to identify new inducers of mitochondrial biogenesis. We hypothesize that screening a large, diverse chemical library with our assay will result in the identification of new chemotypes that produce mitochondrial biogenesis, leading to new chemical entities that produce mitochondrial biogenesis in vivo. In Phase I of the project we propose to screen the DIVERSet 50,000 compound library. The library will be initially screened as pools of five compounds (Aim 1) followed by deconvolution to single molecules (Aim 2), and finally validation of the hits using biochemical assays for mitochondrial proteins and DNA (Aim 3). In Phase II, we will identify chemotypes using in silico pharmacophore analyses and QSAR, synthesize analogues to improve activity, and determine efficacy and potency of the best analogues in inducing mitochondrial biogenesis in RPTC and in mice. ) PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is a major contributor to acute tissue injuries and chronic diseases. At this time, there are no effective therapies that can restore mitochondrial function to promote cell and organ repair/regeneration after diverse acute and chronic injuries. Thus, research is needed to develop new drugs that promote mitochondrial biogenesis.